The function of the lampbrush loops formed by the Y chromosome of Drosophila hydei in spermatocyte nuclei

Summary The function of pairs of translocated fragments of the Y chromosome of Drosophila hydei was tested. As the pairs of fragments together had a complete set of Y chromosomal sites, complementation of their function could be predicted according to results of earlier experiments. In contrast to the earlier experiments the development of lampbrush loops during the spermatocyte stage was blocked in one partner of each combined pair. As a consequence, no complementary effect on spermiogenesis is detectable. The results indicate that the formation of lampbrush loops by seven sites in the Y chromosome is a necessary prerequisite for the normal progress of spermiogenesis. This can be considered as further support of the view that the lampbrush loops in spermatocyte nuclei of Drosophila are phenotypic manifestations of the activity of male fertility factors.Supported by the Deutsche Forschungsgemeinschaft.     

The splicing of transposable elements and its role in intron evolution

Recent studies have demonstrated that transposable elements in maize and Drosophila are spliced from pre-mRNA. These transposable element introns represent the first examples of recent addition of introns into nuclear genes. The eight reported examples of transposable element splicing include members of the maize Ac/Ds and Spm/dSpm and the Drosophila P and 412 element families. The details of the splicing of these transposable elements and their relevance to models of intron origin are discussed.     

Evolution of Y chromosomal lampbrush loop DNA sequences of Drosophila

The evolutionary conservation of Y chromosomal DNA sequences of Drosophila hydei in different species of the genus Drosophila was studied by in situ hybridization and on genomic DNA blots of restriction enzyme digested DNA. We demonstrated that Y specific DNA sequences, which form major parts of lampbrush loops related to the male fertility genes, are only retained in a few closely related species during evolution. Other Y chromosomal DNA sequences, also present in lampbrush loops but with homology to autosomal and X chromosomal locations, were found in distant species. We propose a model for the evolution of the Y chromosomal lampbrush loops.     

Colonization of heterochromatic genes by transposable elements in Drosophila

As a further step toward understanding transposable element-host genome interactions, we investigated the molecular anatomy of introns from five heterochromatic and 22 euchromatic protein-coding genes of Drosophila melanogaster. A total of 79 kb of intronic sequences from heterochromatic genes and 355 kb of intronic sequences from euchromatic genes have been used in Blast searches against Drosophila transposable elements (TEs). The results show that TE-homologous sequences belonging to 19 different families represent about 50% of intronic DNA from heterochromatic genes. In contrast, only 0.1% of the euchromatic intron DNA exhibits homology to known TEs. Intraspecific and interspecific size polymorphisms of introns were found, which are likely to be associated with changes in TE-related sequences. Together, the enrichment in TEs and the apparent dynamic state of heterochromatic introns suggest that TEs contribute significantly to the evolution of genes located in heterochromatin.     

Mammalian Male Mutation Bias: Impacts of Generation Time and Regional Variation in Substitution Rates

In mammals, males undergo a greater number of germline cell divisions compared with females. Thus, the male germline accumulates more DNA replication errors, which result in male mutation bias—a higher mutation rate for males than for females. The phenomenon of male mutation bias has been investigated mostly for rodents and primates, however, it has not been studied in detail for other mammalian orders. Here we sequenced and analyzed five introns of three genes (DBX/DBY, UTX/UTY, and ZFX/ZFY) homologous between X and Y chromosomes in several species of perissodactyls (horses and rhinos) and of primates. Male mutation bias was evident: substitution rate was higher for a Y chromosome intron than for its X chromosome homologue for all five intron pairs studied. Substitution rates varied regionally among introns sequenced on the same chromosome and this variation influenced male mutation bias inferred from each intron pair. Interestingly, we observed a positive correlation in substitution rates between homologous X and homologous Y introns as well as between orthologous primate and perissodactyl introns. The male-to-female mutation rate ratio estimated from concatenated sequences of five perissodactyl introns was 3.88 (95% CI = 2.90–6.07). Using the data generated here and estimates available in the literature, we compared male mutation bias among several mammalian orders. We conclude that male mutation bias is significantly higher for organisms with long generation times (primates, perissodactyls, and felids) than for organisms with short generation times (e.g., rodents) since the former undergo a greater number of male germline cell divisions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Deborah Charlesworth]     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.     

Genes controlling chromosome activity

Normal propagation of Y chromosome lampbrush loops was used as a screening tool in order to recover X-linked mutations controling Y chromosome activation. The nature of the most extreme mutationthus recovered, sterile (1) XL2, is described. It is a recessive gene mutation, readily mapped 2 cross over units distally to white. The mutation exerts its sterilizing effect by blocking normal unfolding of all Y lampbrush loops, but does not affect the unique shape of each diminutive loop. The degree to which a loop forming site is developed is partially temperature sensitive. It is independent however, on its map location or the dose of homologous as well as heterologous sites. It was provisionally concluded therefore that site response to the XL2 effect is a stage specific and not a quantitative one. The possible ways by which non homologous genes control Y chromosome activity are discussed.     

Characterization of Maltase Clusters in the Genus <Emphasis Type="Italic">Drosophila</Emphasis>

To reveal evolutionary history of maltase gene family in the genus Drosophila, we undertook a bioinformatics study of maltase genes from available genomes of 12 Drosophila species. Molecular evolution of a closely related glycoside hydrolase, the α-amylase, in Drosophila has been extensively studied for a long time. The α-amylases were even used as a model of evolution of multigene families. On the other hand, maltase, i.e., the α-glucosidase, got only scarce attention. In this study, we, therefore, investigated spatial organization of the maltase genes in Drosophila genomes, compared the amino acid sequences of the encoded enzymes and analyzed the intron/exon composition of orthologous genes. We found that the Drosophila maltases are more numerous than previously thought (ten instead of three genes) and are localized in two clusters on two chromosomes (2L and 2R). To elucidate the approximate time line of evolution of the clusters, we estimated the order and dated duplication of all the 10 genes. Both clusters are the result of ancient series of subsequent duplication events, which took place from 352 to 61 million years ago, i.e., well before speciation to extant Drosophila species. Also observed was a remarkable intron/exon composition diversity of particular maltase genes of these clusters, probably a result of independent intron loss after duplication of intron-rich gene ancestor, which emerged well before speciation in a common ancestor of all extant Drosophila species.     

Temperatursensitive Mutanten im Y-chromosom von Drosophila hydei

Mutations were induced in the Y chromosomal fertility genes of Drosophila hydei by EMS treatment of adult males. Four types of mutants were observed: 1. Sterile mutants without detectable cytological changes in Y chromosomal lampbrush loops. 2. Sterile males with morphologically changed loops. 3. Sterile males where one or several Y chromosomal loops are missing in the spermatocytes. 4. Mutants which are temperature-sensitive for sterility, development of loops or altered loop morphology. In this paper four Y mutants are described which are temperature-sensitive as regards fertility but which show unchanged lampbrush loops. They can be mapped in four different complementation groups. Two of those occur probably in regions of the Y chromosome without cytologically detectable lampbrush loops. All mutations are found in the distal half of the long arm. The temperature-sensitive period occurs during the primary spermatocyte stage and in early spermatid development while the manifestation of the effect occurs postmeiotically. The mutants are further characterized with respect to changes in the ultrastructure of the sperm at the restrictive temperature.     

Common Mechanisms of Y Chromosome Evolution

Y chromosome evolution is characterized by the expansion of genetic inertness along the Y chromosome and changes in the chromosome structure, especially the tendency of becoming heterochromatic. It is generally assumed that the sex chromosome pair has developed from a pair of homologues. In an evolutionary process the proto-Y-chromosome, with a very short differential segment, develops in its final stage into a completely heterochromatic and to a great extends genetically eroded Y chromosome. The constraints evolving the Y chromosome have been the objects of speculation since the discovery of sex chromosomes. Several models have been suggested. We use the exceptional situation of the in Drosophila mirandato analyze the molecular process in progress involved in Y chromosome evolution. We suggest that the first steps in the switch from a euchromatic proto-Y-chromosome into a completely heterochromatic Y chromosome are driven by the accumulation of transposable elements, especially retrotransposons inserted along the evolving nonrecombining part of the Y chromosome. In this evolutionary process trapping and accumulation of retrotransposons on the proto-Y-chromosome should lead to conformational changes that are responsible for successive silencing of euchromatic genes, both intact or already mutated ones and eventually transform functionally euchromatic domains into genetically inert heterochromatin. Accumulation of further mutations, deletions, and duplications followed by the evolution and expansion of tandem repetitive sequence motifs of high copy number (satellite sequences) together with a few vital genes for male fertility will then represent the final state of the degenerated Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic function correlated with unfolding of lampbrush loops by the Y chromosome in spermatocytes of Drosophila hydei

Summary Deficiencies of the Y chromosome of Drosophila hydei including sites which develop lampbrush loops invariably cause sterility of males. Suppression of loop unfolding in one or more sites equally results in similar morphogenetic defects of spermiogenesis. A variegated type repression of lampbrush loop unfolding observed during the spermatocyte stage results in varying morphogenetic effects on spermiogenesis. This demonstrates the existence of causal relationships between the active phase of Y chromosomal factors in spermatocytes and the differentiation processes in spermatids.In some translocated Y fragments the mode of unfolding of a particular pair of lampbrush loops may be permanently changed. As a result, lampbrush loops of a mutant phenotype are developed. Some alterations of this type are correlated with functional alterations resulting in defective spermiogenesis.Three different fragments of the Y chromosome in which lampbrush loop formation was repressed have been tested for possible reversions of loop suppression by means of X irradiations. In none of the three cases reversion has been detected among two thousand tested chromosomes.To the memory of Karl-Heinz Bier.     

“Hybrid” X-Y translocation chromosomes of Drosophila hydei and D. neohydei

The Y chromosome of Drosophila carries fertility genes which, in part, develop lampbrush loops during the meiotic prophase. Hybrid males from crosses between D. hydei and D. neohydei are fertile although the morphology of the lampbrush loops differs between both species. With the aid of X ray induced hybrid X – Y translocation chromosomes the question has been studied whether Y chromosomal genes of D. neohydei can substitute deletions in the Y chromosome of D. hydei. Although the induction of translocation chromosomes almost regularly results in an inactivation of the translocated Y fragment within a few generations, one case of successful complementation has been demonstrated. Furthermore, a new lampbrush loop pair has been detected in D. neohydei which is morphologically similar to the nooses of D. hydei. Preliminary evidence for the location of the lampbrush loops on the Y chromosome of D. neohydei is discussed.     

Spermatogenesis inDrosophila hydei: A genetic survey

Summary We constructed balancer-chromosomes for the large autosomes ofDrosophila hydei and screened more than 16000 chromosomes for male sterile mutations in order to dissect spermatogenesis genetically. 365 mutants on the X chromosome and the autosomes 2, 3, and 4 were recovered and analysed cytologically in squash preparations under phase-contrast optics. The majority of the mutations allows a rather advanced differentiation of the spermatozoa. At the light-microscopical level, it is possible to classify these mutations with respect to individualization, coiling or motility of the mutant spermatozoa. In contrast, a small number of mutants exhibits conspicuous, pleiotropic phenotypes. Gonial divisions, the shaping of the spermatocyte nucleus and male meiotic divisions are controlled by X chromosomal or autosomal genes which can mutate to male sterile alleles. A number of nonallelic 3^rd chromosome male sterile mutations interfere with the unfolding of the Y chromosomal lampbrush loops. Other autosomal male sterile mutations modify the morphology of these lampbrush loops. Another group of mutations inhibits the formation of the nebenkern while the development of the spermatid nucleus and the flagellum can proceed. Such male sterile mutations can decouple the development of nucleus, protein body, nebenkern, and flagellum of the spermatid. Thus, we can describe spermatogenesis inDrosophila as the coordinate execution of the individual developmental programs of the different components of the spermatozoon.     

Autosomal control of lampbrush-loop formation during spermatogenesis in Drosophila hydei by a gene also affecting somatic sex determination

Summary The Y chromosome of Drosophila hydei carries information that is necessary for the development of the spermatozoa. In primary spermatocytes Y chromosomal genes become active: five of the male fertility factors form giant lampbrush loops. Our prior work indicated interactions between the Y chromosomal genes and autosomal loci. It is of interest to identify loci regulating the activity of the Y chromosomal genes. We, therefore, screened a total of about 14,000 chromosomes (X, 2, 3 and 4) for mutations that interfere with the expression of the lampbrush loops. Two mutations with substantial effects on the loop morphology were recovered. One of them, a recessive male sterile mutation (ms (3) 5) on chromosome 3, is described in this paper. Its homozygous state results in a complete absence of all Y chromosomal lampbrush loops at 26° C; at 18° C the loops are formed. Temperature shifts with homozygous males indicate that the function early during the spermatogonial stage is crucial for the development of lampbrush loops in the primary spermatocyte. Meiosis is entirely absent in the male, but normal in females. Females homozygous for ms (3) 5 display a maternal effect, which reduces the viability and fertility of homozygous daughters and produces sons with signs of intersexuality. Linkage studies indicated that the effect on the male germ line and the maternal effects cannot be separated and may hence be induced by a single gene.